Enveloped viruses infect target host cells through protein mediated membrane fusion. Influenza hemagglutinin (HA) has served as the paradigm for understanding the general mechanism of membrane fusion and it is also an important target for antiviral drug development. The central hypothesis of the proposal is that pH induced refolding of hemagglutinin drives the fusion of the viral and host membranes. The mechanism of membrane fusion has been inferred from equilibrium structures of fragments of hemagglutinin, along with biochemical evidence. Hemagglutinin is postulated to undergo an astounding series of refolding reactions triggered by lowered pH, first to form an extended coiled coil conformation that exposes the fusion peptides and then an even more dramatic refolding to an antiparallel coiled-coil, six-helix bundle scaffold. Finally, a zipping of linker domains against this scaffold s postulated to drive the membranes together and facilitate membrane fusion. We plan to elucidate the molecular details of this dynamic refolding process, and the coupled interactions with lipid bilayers that accomplish membrane fusion, using time-resolved spectroscopic methods that we have developed to study protein folding in membranes. Our approach will focus on all of the critical components of the complex mechanism, including the pH induced formation of an extended coiled-coil by the trigger peptide region (L40) and the insertion of the fusion peptide into the host membrane to start the fusion process. We will study these two functional units in the soluble HA2 domain to determine how these processes are coupled. We will also determine if the complete soluble hemagglutinin protein refolds to form an antiparallel coiled-coil structure coupled to these earlier steps, followed by the refolding of the linker domains against this scaffold. These studies will make use of laser induced pH and temperature jump methods pioneered in our laboratory, as well as ultrafast mixing to rapidly initiate the HA refolding reaction, and time resolved IR and fluorescence spectroscopy to follow the dynamics with high structural specificity. We expect that our unique approach combined with our focus on HA as an archetype will provide an unprecedented molecular view of the dynamic function of this important class of protein machines, and thereby improve our understanding of protein mediated membrane fusion. More generally, we expect a better understanding of the dynamics and molecular mechanisms of protein folding in membranes to emerge from this work. Protein folding at the boundary of or within the membrane is a process that has been very difficult to study and as a consequence is poorly understood. As a basis for understanding the dynamic interactions of HA with the lipid bilayer, we propose to study fundamental folding processes of model systems at the interface of or within membranes, including membrane association, insertion, folding, and assembly into higher order structures.